Cytochrome P450

If you want to understand how different people vary in their response
to drugs and toxins (xenobiotics) that have entered their body,
you have to know something about cytochrome P450!
A core knowledge of CYP is vital if you wish to anticipate
potentially lethal drug interactions. It is possible that doctors in
the USA alone may kill about one hundred thousand people per year
through drug interactions! We have
no indication that even in more civilized countries, this figure is
lower.

This Web-page is intended as an introduction to CYP and its
importance in clinical medicine. We look at:

1. What is CYP?

'CYP' is a host of enzymes that use iron to oxidise things,
often as part of the body's strategy to dispose of potentially
harmful substances by making them more water-soluble. Bertz
and Granneman (Clin Pharmacokinet 1997 32 210-58) found that 56%
of 315 drugs were primarily cleared by CYP! Adding something like a
hydroxyl group to a xenobiotic is just part of the body's strategy to get
rid of the 'drug' - this is often followed by conjugugation to groups such as
glucuronide to increase the solubility even further. To try and
thoroughly confuse you, the initial P450-mediated oxidation is often
referred to as "Phase I metabolism" and the subsequent conjugation
(which has nothing to do with P450) as "Phase II".

Where is it found?

It is not surprising that much of the CYP in man is found in the liver,
the main organ involved in drug and toxin removal, but a remarkable
amount is also found in the small intestine. CYP usually sits around in
the 'microsomal' part of the cytoplasm (endoplasmic reticulum). Metabolic
clearance of drugs is not the only function of CYP - recently, it has been
found that CYP is intimately involved in vascular autoregulation, particularly
in the brain. CYP is vital to the formation of cholesterol, steroids
and arachidonic acid metabolites. Other functions surely remain to be uncovered.

How many different CYPs are there?

There are over a thousand different CYPs, although the number in man
is only about fifty (49 genes and 15
pseudogenes have been sequenced). It is
likely that most of the human CYPs have already been discovered.
Why are there so many varieties of CYP? The massive hetereogeneity
of these oxidases is thought to reflect the complex interdependence
{read: 'ongoing battle'} between plants and animals. Plants develop
new alkaloids to limit their consumption by animals - the
animals develop new enzymes to metabolise the plant toxins, and so it goes.
It is possible to peer back in evolution by looking at similarities
between CYP isoenzymes.
When we do so, it appears that
the number of CYP genes exploded at about the time when organisms moved
from the oceans to dry land - around 400 million years ago!

What's in a name?

First-time readers may wish to skip the
following section, as it may well confuse them. Click
here to skip over our exploration
of the peculiar origins of the name of cytochrome P450!

Why cytochrome P 450? There's a story attached to this.
Initially, when researchers
realised how important cytochromes were in
metabolism, they needed a way of identifying them unequivocally. We know
that most CYP is anchored to membranes of the microsomal portion of the cell. This
attachment is unfortunate for investigators, as grinding up cells and
extracting the microsomal portion results in a rather opaque suspension.
Special tricks are needed to identify the CYP component - the microsome-containing
solution is divided into two (after adding an agent that reduces any haem that might be present),
and one part is exposed to carbon monoxide.
If the solution exposed to CO strongly absorbs light at a wavelength of
450nm compared with the original solution, it must contain CYP.
This is called "difference spectroscopy", and we are finding the
"reduced CO difference spectrum". (The P in P450 stands for "pigment").

{Fine Print:
When we said that absorption at 450nm on exposure to CO uniquely identifies P450,
we lied just a little. The reason why absorption occurs at this wavelength
is related to one of the six 'ligands' associated with the iron atom contained in
the haem. The haem ring itself provides four ligands (nitrogens), but in P450
the fifth is an unusual, negatively charged sulphur atom. This is known
to its few friends as a "thiolate anion", and proteins containing this
unusual moiety are called "haem-thiolate proteins". There are
other haem-thiolate proteins apart from P450 - they include
cystathionine beta-synthetase (EC 4.2.1.22), haem chloroperoxidase
(1.11.1.10) and nitric oxide synthetase (1.14.13.39).

Cytochrome P450 chemistry is fascinating and challenging. Note that
the bond between the two atoms in an oxygen molecule is rather strong.
This implies that a substantial amount of energy is required to break
the bond - energy that is supplied by addition of electrons to the
iron atom of heme. These electrons in turn come from the last protein in
an "electron transfer chain". There are two such chains in cells that end
up at P450. The first is in the endoplasmic reticulum (ER), and the
protein involved is called NADPH cytochrome P450 reductase - electrons
pass from NADPH to FAD to FMN and thence to heme. The second
chain lurks within mitochondria. A complex bucket brigade of proteins
hands the electrons down to heme. NADPH passes electrons to ferredoxin
reductase, thence to ferredoxin (which itself has an iron-sulphur cluster),
and from there to CYP.

}

2. CYP Isoforms

This section looks at how we classify CYP, polymorphism and its
importance, and enzyme induction as
well as other controversial issues such as the importance of CYP to drug
design, the relationship between CYP and P-glycoprotein, and how CYP
has been implicated in causing cancer and other diseases.

How do we classify CYP?

There are numerous isoforms of cytochrome P450. (An isoform is
a CYP enzyme variant that derives from one particular gene). They are classified
according to the similarities of their amino-acid sequences.
Such classification allows division of CYP isoforms into:

families - CYP families contain genes that have at least a 40%
sequence homology.
There are at least 74 CYP families, but only about seventeen of these
have been described in man.

subfamilies - Members of a subfamily must have at least a 55%
identity.
About thirty subfamilies are well characterised in man

individual genes - There are fifty or so genes important in man.

Families are numbered - for example CYP2, CYP21.
Subfamilies
are identified by a letter, and thus we get CYP3A, CYP2D.
Individual
genes are identified by a number, for example CYP2D6.

Among the diverse human genes, several have been identified as
particularly important in oxidative metabolism. They are:

Other notable CYPs are CYP2E1,
CYP2A6, and
CYP1A2. On exposure to
appropriate substrates, enzyme induction occurs with all of
these CYPs, apart from CYP2D6. In addition, those in italics
above are polymorphic.

What is polymorphism?

In different people and different
populations, activity of CYP oxidases differs.
Genetic variation in a population is termed 'polymorphism' when
both gene variants exist with a frequency of at least one percent.
Such differences in activity
may have profound clinical consequences, especially when multiple drugs
are given to a patient. There are profound racial differences in the
distribution of various alleles - data on a drug that works in one way in
one population group cannot necessarily be extrapolated to another group.

Why does polymorphism occur, and why is it important?

The explanations for the various polymorphisms are thought to be complex, but
perhaps the most interesting is the high expression of CYP2D6 in many
persons of Ethiopian and Saudi Arabian origin. 2D6 is not inducible, so these people have
developed a different strategy to cope with the (presumed) high load of
toxic alkaloids in their diet - multiple copies of the gene.
These CYPs therefore chew up a variety of drugs, making them ineffective -
many antidepressants and neuroleptics are an important example. Conversely,
prodrugs will be extensively activated -
codeine will be turned in vast amounts into morphine!

In contrast, many individuals lack functional 2D6. These subjects will
be predisposed to drug toxicity caused by antidepressants or neuroleptics,
but will find codeine (and indeed, tramadol) to be inefficacious due to
lack of activation! Other drugs that have caused
problems in those lacking 2D6 include dexfenfluramine, propafenone,
mexiletine, and perhexiline. Perhexiline was in fact withdrawn from the market due to
the neuropathy it caused in those 2D6 inactive patients unfortunate enough
to be treated with it. Even beta-blocker removal may be impaired (for example,
propranolol) in 2D6-deficient people.

Another potentially disastrous polymorphism is deficient activity
of CYP2C9. This is because patients possessing this enzyme variant are ineffective in
clearing (S)-warfarin - so much so that they may be fully
anticoagulated on just 0.5mg of warfarin a day! As if this isn't enough,
the same CYP is important in removal of phenytoin and tolbutamide, both
potentially very toxic drugs in excess. The flip-side is that the
prodrug losartan will be poorly activated and inefficacious with 2C9
deficiency. Azole antifungals, sulphinpyrazone, and even amiodarone may
cause a similar effect by inhibiting the enzyme.

Occasionally one derives benefit from an unusual CYP phenotype.
For example, cure rates for peptic ulcer treated with omeprazole are
substantially greater in individuals with defective CYP2C19, owing to
the sustained, high plasma levels achieved.

Is enzyme induction of CYP important?

Yes. Although most of the CYPs can be induced (the notable exception being 2D6),
perhaps the most important in this regard is CYP3A4. 3A4 is the most
prevalent CYP in the body, and metabolises many substrates. The most important
inducers of 3A4 are antimicrobials such as rifampicin, and anticonvulsants
like carbamazepine and phenytoin, but potent steroids such as
dexamethasone may also induce 3A4. The long list of agents metabolised by
the enzyme include opioids, benzodiazepines and local anaesthetics,
as well as erythromycin, cyclosporine, haloperidol, calcium channel blockers,
cisapride and pimozide. Oral contraceptives are also metabolised, and their
efficacy may be impaired when an inducer such as rifampicin is taken.

Even more important than the inducers of 3A4 are the inhibitors.
There is a long list - azole antifungals, HIV protease inhibitors, calcium
channel blockers, some macrolides like troleandromycin and erythromycin,
and the commonly used 'SSRI' antidepressants. Lethal clinical consequences
can result from combining 3A4 inhibitors with drugs that are metabolised
by this cytochrome. Non-sedating antihistamines have resulted in fatal
arrhythmias, as has occurred with cisapride administration in combination
with an inhibitor. Erythromycin in combination with theophylline may cause
toxicity due to the latter.

Where does P-Glycoprotein come in?

There is an interesting association between some CYPs and the important
transmembrane pump protein, P-glycoprotein (the product of the MDR1 gene).
Generally, if P-glycoprotein is there, then CYP3A4 is not far behind.
This seems to be part of a concerted strategy by the body to eliminate
xenobiotics - the P-glycoprotein pumps out what it can, and CYP3A zaps
the rest! This association makes for even more interesting drug
interactions, for example calcium-channel blockers interact with
the membrane pump and the CYP! The same holds for drugs as diverse
as azole antifungals, immunosuppressants and macrolides.

Does CYP have something to tell us about drug design?

Designing and ultimately marketing a drug costs a bomb. The interaction
between CYP and freshly minted drugs is therefore rather important to
pharmaceutical companies, so much so that predominant degradation of a drug by
one of the polymorphic CYPs is often enough to stop further research
on that drug in its tracks!

Is CYP important in cancer?

There has been much speculation about the role of the various CYP proteins
and polymorphisms as causes of cancer. Some CYPs may activate pro-carcinogens
to carcinogens; many are probably involved in the removal of carcinogens
from the body. In addition, several cancers are hormone sensitive, and
those CYPs involved in, for example, steroid or retinoic acid metabolism
may play a crucial role in suppression or promotion of malignancies through
such metabolism.

There has been much speculation (but little production of hard evidence)
that some CYPs found in the lung promote lung cancer, especially in
cigarette smokers.

Which CYPs localise in specific tissues?

Some isoforms are found throughout the body, for example CYP51,
while others are limited to one specific tissue (take CYP11B2,
found mainly if not exclusively in the glomerulosa zone of the
adrenal gland)!

Differential expression of some CYPs in different organs may
also have clinical consequences, especially where the unfortunate side-effect
of 'degradation' of a drug is to make a more toxic product. The degradation
of paracetamol by 2E1 results in a highly active intermediate product
which in sufficient quantities can result in fulminant liver failure.
Anti-oxidants protect against this catastrophe; in contrast, chronic
ethanol consumption induces 2E1 and may increase the likelihood of toxicity.

Does CYP change with age?

Variable expression of CYP has substantial clinical consequences,
not only in different people and different race groups, but also
in individuals as they progress from infancy to old age. For example:
CYP1A2 is not expressed in neonates, making them particularly susceptible
to toxicity from drugs such as caffeine.

3. Clinical Nitty Gritty

The clinical consequences of CYP polymorphism, inhibition and inducibility
have already been mentioned. Unfortunately, these are potentially so complex
that whenever you give a drug you should ask whether it has an effect on
a CYP isoform, and whether it is metabolised by CYP.

Here are just some reminders. Note that many of the following interactions
and other data are from single studies, or have been read only in abstract.
Do NOT apply the following data to patients. Instead, consult other
(preferably human) sources. If there is no apparent
reference for an assertion, the reference is often included as a comment
in the html source! (This is to decrease the
apparent clumsiness of the pages - Click View | Source in your browser).

Drugs and CYP - Tables

Drug name starts with..(Click on letter to enter table)

Examples(If the drug you want isn't in this column, still look for it in
the table corresponding to the first letter of its generic name!)

4. A Table of all the isoforms

The following table is meant to reference all the known CYP isoforms in man.
Click on any one of the families to view details.
Remember, that things change practically on a daily basis, so do NOT
regard the following as authoritative, especially in terms of drug
interactions. Do NOT base clinical decisions on the following tables.

CYP PseudogenesThere are 15 known
(a pseudogene is a gene relic that is not expressed in
normal tissues)

CYP2A7 (2A7PT, 2A7PC)

CYP2B7P

CYP2D7AP

CYP2D8P, 2D8BP

CYP2F1P

CYP2 : 1 other pseudogene ???

CYP3A5P1 and 3A5P2 : 2 pseudogenes

CYP4F9P

CYP4F10P

CYP21A1P

CYP51 : 2 pseudogenes (CYP51P1, CYP51P2)

Dubious CYP genesThe following genes either don't exist, are poorly characterised,
or are thought not to play a role in human metabolism
(see also pseudogenes above)

CYP2C10

?

CYP2C11

!

CYP3A3

an artefact

CYP4A9

artefact

5. References

An excellent Cytochrome P450 webpage is
David Nelson's.
We have already mentioned
David Flockhart and
Ed Hayes' pages.
An important reference on different alleles is
the Human Cytochrome P450 (CYP) Allele Nomenclature Committee's
web page.
The
CYP page of Kirill N. Degtyarenko and Péter Fábián contains a wealth of
information and links. Visit it!
Much of the above page was derived
from these and the following articles: